Generally, the present invention relates to the discovery and utilization of a biological oxygen sensor in the diagnosis, prognosis, prevention and/or treatment of undesirable biological states. More particularly, the present invention relates to improving or restoring cellular and/or tissue oxygenation, preferably to benefit undesirable states associated with tissue oxygen sensing. The present invention generally contemplates devices, drugs, or treatment methodologies involved with tissue oxygenation, especially in tissue that would benefit from such treatment. The methodologies contemplated herein include gene therapy, protein replacement therapy, and protein mimetics.
It is postulated that as certain tumors enlarge, tissue often outgrows its oxygen and nutrient supply because of an inadequate network of functioning blood vessels and capillaries. Although cells deprived of oxygen and nutrients may ultimately die, at any given time a tumor cell may produce viable hypoxic cells that are viable in hypoxic environments. Hypoxic cells, although alive, have very low oxygen concentrations because of their remoteness from the blood vessels.
Hypoxia appears to contribute to resistance to radiotherapy and chemotherapy in many tumors. Hypoxia can also be an independent poor prognostic factor in many cancers, e.g. cervical cancer. Hypoxic stimulation provides environmental pressure for further tumor cell selection, growth and progression to more aggressive stages. This is referred to as hypoxia-mediated selection. For example, p53 mutations found in solid cancers, are proposed to be the result of such hypoxia-mediated selection. Mutations in p53 are postulated to confer resistance to apoptosis, a cellular defense mechanism that leads to cell death.
Hypoxic conditions increase a tumor""s resistance to conventional treatment in a number of ways. When chemotherapeutic agents are administered to patients, the agents are carried through the functioning blood vessels and capillaries to the target tissue. Because hypoxic tissue lacks a fully functioning blood supply network, chemotherapeutic drugs may never reach the hypoxic cells; instead, intervening cells scavenge the drug. The result is that the hypoxic cells survive and recurrence of the tumor is possible.
Hypoxia also hinders the effectiveness of radiation therapy, especially of neoplasms. Radiation treatment is most effective in destroying oxygen containing cells because oxygen is an excellent radiation sensitizer. The presence of hypoxic cells impedes this treatment. Therefore, hypoxic cells are more likely to survive radiation therapy and eventually lead to the reappearance of the tumor. The importance of hypoxic cells in limiting radiation responsiveness in animal tumors is well known. In addition to its role in tumorigenesis, hypoxia is a factor in the pathogenesis of major causes of mortality including myocardial ischemia, stroke, cancer, and chronic lung disease. Hypoxia plays a role in many diverse disease states.
One of the most important goals in oncology is the identification and elimination of treatment resistant cells; hypoxic cells being the most familiar examples of this type of cell. Surprisingly the carotid body in mammals may provide insight into these treatment resistant cells. In mammals, the carotid body (CB) appears to play a major role in acute adaptation to hypoxia by stimulating the cardiopulmonary system. Chronic exposure of mammals to hypoxic conditions, observed for example in humans living at high altitudes, induces hyperplastic/anaplastic growth in the carotid body. Similar hyperplastic/anaplastic growth in the CB is observed in individuals with chronic arterial hypoxemia, such as cyanotic heart and chronic lung diseases. The carotid body is a highly vascular small organ located at the bifurcation of the common carotid artery in the neck and is a chemoreceptive organ involved in sensing oxygen levels in the blood.
Stimulation of the cardiopulmonary system results in increased blood flow and subsequent increase in oxygenation of tissue. At the cellular level, this sequence is thought to involve a hypoxia-inducible transcription factor (HIF-1), activation of which leads to a systemic response. The systemic response includes an increase in red cell mass, stimulation of new blood vessel growth, and increased ventilation.
As will be more fully described herein, it appears that the mitochondrial complex II cytochrome b is an important (potentially primary) oxygen sensor in mammalian cells. The protein subunits of mitochondria complex II cytochrome b appears to be involved in oxygen sensing and provide a novel genetic and pharmacological target. Therapies designed to amplify adaptive responses to hypoxia and, conversely, to inhibit these responses in cancer cells are now viable. A comprehensive understanding of the mitochondrial complex II cytochrome b also provides a target for effective therapies for neurodegenerative diseases such as Parkinson""s and Alzheimer""s.
One aspect of the present invention is a method for treating diseased states especially in mammals. These diseased states are preferably associated directly or indirectly with tissue oxygenation. Accordingly, one embodiment of the present invention describes a method of treating a diseased state in a human comprised of identifying hypoxic cells, and supplying an oxygen sensor to said cells. It may also be preferable to create a hyperbaric environment for the cells to which the oxygen sensor is provided.
The present invention further provides methods for correcting and/or augmenting oxygen sensing defects in cells. Preferred methods involve supplying the SDHD gene or cybS protein, or any of the other components of the mitochondrial complex II cytochrome b oxygen sensing complex in an amount which restores or imposes oxygen sensing to affected or surrounding cells, and thus facilitates normoxic conditions.
An additional aspect of the invention relates to creating hypoxic cells by incorporating missense or nonsense mutations in an SDHD gene and supplying such a mutated SDHD gene to desired cells.
Further, the invention relates to the PGL1 locus, cloning vectors, expression systems and recombinant cells comprising the SDHD coding sequence, the PGL1 locus, and variations thereof.
A further embodiment of the present invention provides nucleic acid probes complementary to the SDHD gene as well as to SDHD mutants. A related aspect of the invention includes DNA primers for amplification of nucleic acid fragments of the PGL1 locus.
Additionally, the present invention provides methods and kits for diagnosing diseased states in mammals including PGL, and those relating to decreased tissue oxygenation or oxygen stress.
An alternative embodiment of the present invention provides a method of treating a diseased state preferably a nuerodegenerative disease in a human by supplying autologous carotid body cells to oxidatively stressed regions.
Yet another feature of the present invention resides in an expression system which includes a DNA sequence, said DNA sequence corresponding to a portion of PGL1 locus. The expression system is a recombinant host cell transformed with said DNA sequence. The portion of PGLL locus corresponds to SEQ ID NO:1. Alternatively, the portion of PGL1 locus corresponds to SEQ ID NO:2. The DNA sequence includes a nucleotide analog which is incapable of being methylated.
Another aspect of the present invention resides in an isolated DNA sequence comprised of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and portions thereof, wherein the sequence includes a mutation selected from the group consisting of nonsense, missense and deletion mutation Alternatively, the mutation is a mutation in SEQ ID NO:1 selected from the group consisting of: a T at nucleotide base number 106 of SEQ ID NO:1; a T at nucleotide base number 112 of SEQ ID NO:1; a T at nucleotide base number 242 of SEQ ID NO:1; and a T at nucleotide base number 305 of SEQ ID NO:1.
Yet another feature of the present invention resides in a method of treating a diseased state comprised of supplying a biological oxygen sensor to a patient in need thereof. The biological sensor is a mitochondrial complex II protein. The mitochondrial complex II protein include a functional cybS subunit. The biological oxygen sensor includes a protein sequence corresponding to SEQ ID NO:3. The biological mitochondrial complex II protein includes a protein sequence corresponding to SEQ ID NO:3. The step of supplying a biological oxygen sensor includes supplying autologous carotid body cells to said patient. The disease may be a neurodegenerative disease. The biological oxygen sensor may be supplied in conjunction with a therapeutic treatment, said treatment being selected from the group consisting of radiation and chemotherapy. The biological oxygen sensor may be resistant to methylation.
The present invention broadly facilitates a number of useful treatments involving oxygen sensing or tissue oxygenation. The treatment may involve identifying hypoxic cells; supplying or restoring a biological oxygen sensor to the cells; and creating a hyperbaric environment for said cells having an oxygen sensor to thereby improve tissue oxygenation.
In one embodiment, the present invention utilizes the proliferative and oxygen sensing capabilities of CB cells by measuring the levels of mitochondrial complex II cytochrome b components (e.g. cybS or cybL. Such measurements may be useful in treating and monitoring patients with neurodegenerative diseases.
Under some circumstances it may be desirable to provide a method of treating a diseased state in an individual, comprising supplying a demethylating agent to tissue in need of such and agent, such that tissue oxygenation is improved. The present invention facilitates such a method, as well as an expression system wherein the isolated DNA contains a non-methylatable nucleotide analog. Further, recombinant host cells transformed with the above expression system are also provided by the present invention.
In addition to providing general treatments for diseased states, the present invention provides a method of augmenting a treatment by identifying hypoxic cells; supplying an oxygen sensor to said cells; and creating a hyperbaric environment for said cells having an oxygen sensor. Examples of the augmented treatment include chemotherapy, radiation therapy, tissue oxygenation, and supplying an oxygen sensor to said cells. Furthermore, these treatments may include the step of creating a hyperbaric environment for said cells having an oxygen sensor.
The present invention also involves the isolated DNAs sequences utilized in a replicative cloning vector which comprises the isolated DNA of the PGL1 locus, SEQ ID NO:1, or SEQ ID NO:2 and a replicon operative in a host cell. Additional embodiments include an expression system which comprises the isolated DNA of the PGL1 locus, SEQ ID NO:1 or SEQ ID NO:2, operably linked to suitable control sequences. Recombinant host cells can be transformed with the any of these replicative cloning vectors.
Various nucleic acid probes and primers of the present invention may also be useful in diagnostic and therapeutic techniques. Among these are a nucleic acid probe complementary to human SDHD gene as well as a nucleic acid probe complementary to human altered SDHD gene sequences wherein said nucleic acid probe hybridizes to a mutant SDHD gene under hybridization conditions which prevent hybridizing of said nucleic acid probe to a wild-type SDHD gene.
The present invention will also facilitate the amplification of segments of the PGL1 locus. Several methods providing for this amplification are described including: a pair of single-stranded DNA primers wherein use of said primers in a polymerase chain reaction results in amplification of an SDHD gene fragment, wherein the sequence of said primers is derived from the regions of the isolated DNA defined by SEQ ID NO:2. Similarly, the invention also provides for a pair of single-stranded DNA primers wherein use of said primers in a polymerase chain reaction results in amplification of an SDHD gene fragment, wherein the sequence of said primers is derived from the exon regions of the isolated DNA defined by SEQ ID NO:1.
In addition to the diagnostic methods detailed above, the present invention also provides kits for detecting SDHD mutations. One such kit for detection of SDHD mutations is one comprising: an oligonucleotide fragment from SEQ ID NO:2; and means for detecting said SDHD nucleic acids. An additional kit detects PGL comprising: a set of primers for detection of SDHD from SEQ ID NO:2; and means for detecting said SDHD nucleic acids.